A novel positive allosteric modulator of the alpha7 neuronal nicotinic acetylcholine receptor: in vitro and in vivo characterization

Abstract

Several lines of evidence suggest a link between the alpha7 neuronal nicotinic acetylcholine receptor (nAChR) and brain disorders including schizophrenia, Alzheimer's disease, and traumatic brain injury. The present work describes a novel molecule, 1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxazol-3-yl)-urea (PNU-120596), which acts as a powerful positive allosteric modulator of the alpha7 nAChR. Discovered in a high-throughput screen, PNU-120596 increased agonist-evoked calcium flux mediated by an engineered variant of the human alpha7 nAChR. Electrophysiology studies confirmed that PNU-120596 increased peak agonist-evoked currents mediated by wild-type receptors and also demonstrated a pronounced prolongation of the evoked response in the continued presence of agonist. In contrast, PNU-120596 produced no detectable change in currents mediated by alpha4beta2, alpha3beta4, and alpha9alpha10 nAChRs. PNU-120596 increased the channel mean open time of alpha7 nAChRs but had no effect on ion selectivity and relatively little, if any, effect on unitary conductance. When applied to acute hippocampal slices, PNU-120596 increased the frequency of ACh-evoked GABAergic postsynaptic currents measured in pyramidal neurons; this effect was suppressed by TTX, suggesting that PNU-120596 modulated the function of alpha7 nAChRs located on the somatodendritic membrane of hippocampal interneurons. Accordingly, PNU-120596 greatly enhanced the ACh-evoked inward currents in these interneurons. Systemic administration of PNU-120596 to rats improved the auditory gating deficit caused by amphetamine, a model proposed to reflect a circuit level disturbance associated with schizophrenia. Together, these results suggest that PNU-120596 represents a new class of molecule that enhances alpha7 nAChR function and thus has the potential to treat psychiatric and neurological disorders.

Figure 1.

PNU-120596 enhances Ca²⁺ flux mediated by an engineered variant of the human α7 nAChR. A, Chemical structure of PNU-120596. B, Plot of the change in relative fluorescence units (ΔRFU) mediated by α7^* expressed in SH-EP1 cells. Cells were pretreated with either vehicle (0.2% DMSO) or 3 μm PNU-120596 (first arrow) and subsequently challenged with either buffer or 100 μm ACh (second arrow). Open circles indicate cells treated first with DMSO and then challenged with buffer, gray squares indicate cells treated first with DMSO and then challenged with ACh, and black diamonds indicate cells treated first with PNU-120596 and then challenged with ACh. C, Concentration-response relationship of PNU-120596 determined from α7^*-expressing SH-EP1 cells (EC₅₀ = 216 ± 64 nm; n = 51). Error bars represent SEM.

Figure 2.

PNU-120596 selectively enhances the function of human α7 nAChRs. A, Typical currents recorded in response to a brief test pulse (3 s) of either choline (1 mm) or ACh (100 μm) in an oocyte expressing the human α7 nAChR. Time course of typical responses recorded before and after 20 s of pretreatment with 1 μm PNU-120596. B, Plot of the mean current amplitude evoked by choline and ACh in the absence and presence of PNU-120596 (1 μm). C, Plot of the average peak current as a function of ACh concentration (n = 4). Currents evoked by a series of ACh pulses were recorded first in control and then after PNU-120596 exposure (same protocolas in A). Data points are the average of three cells, and error bars indicate the SDs. Curves through the data points are the best fit obtained with the empirical Hill equation. The EC₅₀ and n_H values were 33.7 ± 5.3 and 2.2 ± 0.4 μm (n = 4) for control conditions and 3.8 ± 0.5 and 4 ± 0.4 μm (n = 5) in the presence of PNU-120596. D, The effects of PNU-120596 are specific to the α7 subtype of nAChR. Ratios of peak current amplitude expressed as fold change recorded before and after PNU-120596 (1 μm) incubation were measured in another batch of oocytes expressing different human nAChR subtypes. ACh test pulses were 100 μm for α7 (n = 4), 50 μm for α4β2 (n = 4), 50 μm for α3β4 (n = 4), and 10 μm for α9α10 (n = 4). Error bars represent SEM.

Figure 3.

PNU-120596 potentiates endogenous α7 nAChRs of cultured rat hippocampal neurons. Aa, Example of currents evoked by 1 mm ACh alone (top trace) and in the presence of 0.1 μm PNU-120596 (middle trace) or 1 μm PNU-120596 (bottom trace); PNU-120596 was added ∼30 s before ACh in each case. Ab, The same data as in Aa but on an expanded time scale; the current amplitudes have been normalized. The gray trace shows the currents evoked by 1 mm ACh in the absence of PNU-120596. B, Consecutive responses to 1 s challenges with ACh (arrows; 1 mm) repeated once perminute. PNU-120596 was applied continuously for ∼5 min as indicated by the horizontal bar. C, Currents evoked by 100 μm ACh alone (left) and in the presence of 1 μm PNU-120596 (right). Currents recorded in the presence of PNU-120596 were recorded in the absence (black trace) and presence (gray trace) of 10 nm MLA.

Figure 4.

PNU-120596 can activate desensitized α7 nAChRs. Nicotine (100 μm) was applied to cultured rat hippocampal neurons for a duration of 2.5 min. After 30 s of continuous exposure tonicotine, PNU-120596 (1 μm) was coapplied for an additional 2 min. The duration of exposure to nicotine and PNU-120596 is indicated by the black horizontal bars above the top trace. The initial response to nicotine is shown by the bottom trace (gray); the small gray bar indicates the region from the top trace that is expanded in the bottom trace.

Figure 5.

PNU-120596 does not modify the ion selectivity of α7 nAChRs. A, Currents evoked by low ACh concentration (10 μm) were recorded first in control conditions and after exposure to PNU-120596. Application of a voltage ramp during the ACh application allowed the determination of the current-voltage relationship shown in B. Lines through the data points are the best fits obtained using Ohm's Law and a Boltzman equation to mimic the voltage dependency (see Materials and Methods). Parameters for the equation were not statistically different (V_e = 0 mV; α = 0.88; V_o = -21.8 mV) in the two conditions, except for the conductances G = 2.3 nS, G_cte = 0.4 nS in control and G = 13.3 nS, G_cte = 4.5 nS after PNU-120596 exposure. Values are the mean results obtained from six oocytes.

Figure 6.

PNU-120596 increases the open time of α7 nAChRs. Aa, Ba, Examples of nicotine-evoked currents recorded from the same outside-out membrane patch in the absence (Aa) and presence (Ba) of 1 μm PNU-120596. Membrane patches were pulled from GH4-C1 cells expressing the rat α7 nAChR. In this example, nicotine was applied for 1 s when given alone and for 250 ms in the presence of PNU-120596; a shorter application of nicotine was required in the presence of PNU-120596 to capture the complete decay of current. Ab, Bb, Expanded regions of the same data as above. Note the 10-fold difference in the time scale between Ab and Bb.

Figure 7.

PNU-120596 enhances the excitatory actions of ACh on hippocampal interneurons in isolated slices. A, Effect of PNU-120596 on the ACh-evoked current in a stratum radiatum hippocampal interneuron. The voltage-clamp records show the inward current evoked by ACh (0.2 mm) in control conditions (top trace) and after the addition of PNU-120596 (2 μm) to the perfusion solution (bottom trace). The horizontal bar above the current traces indicates the time during which ACh was pressure ejected. The patch pipette was filled with a K-gluconate-containing solution, and the holding potential was -70 mV. B, Continuous recording from pyramidal neurons showing spontaneously occurring GABAergic synaptic activity. After 3 min of baseline measurement, ACh (10 μm), PNU-120596 (0.3 μm), or the combination of ACh (10 μm) and PNU-120596 (0.3 μm) was bath applied for an additional 10 min. C, Plot of the change in synaptic activity relative to the baseline period measured for the three experimental groups. Data points are the average number of events recorded during consecutive 60 s bins for ACh alone (open squares; n = 5), PNU-120596 alone (open circles; n = 5), and ACh plus PNU-120596 (filled circles; n = 6). In each case, baseline activity was measured over a 3 min period, and test solutions were bath applied for 10 min. D, Effect of ACh on spontaneous GABAergic IPSCs in a CA1 pyramidal neuron. All recordings were performed in the presence of PNU-120596 (2 μm). The synaptic activity was recorded in the absence (first and third traces) and in the presence (second and fourth traces) of ACh (50 μm). TTX (1 μm) was present when the third and fourth traces were recorded. Patch pipettes were filled with a CsCl-containing solution, and the holding potential was -70 mV. Note that TTX suppressed the ACh-dependent increase in inhibitory synaptic activity. Error bars represent SEM.

Figure 8.

PNU-120596 improves auditory gating deficits induced by d-amphetamine in anesthetized rats. A, Hippocampal auditory evoked potentials (summation of 50 consecutive evoked potentials, displayed at a continuous 1 s sweep) in response to conditioning and test auditory tones (indicated by dashed lines; intertone interval, 0.5 s) in control conditions (black trace) and after administration of d-amphetamine (gray trace; 1.0 mg/kg, i.v.). B, Typical hippocampal auditory evoked potentials (summation of 50 consecutive evoked potentials) in response to conditioning (black trace) and test (gray trace) auditory tones in control conditions after administration of d-amphetamine (Amph; 1.0 mg/kg, i.v.) and after a subsequent administration of vehicle (PBS) or PNU-120596 (1 mg/kg, i.v.). C, Average of hippocampal auditory gating in rats (expressed as a percentage) during the control period, after d-amphetamine (1.0 mg/kg, i.v.), and after subsequent administration of vehicle (PBS; 1 ml/kg, i.v.; n = 8) or PNU-120596 (1 mg/kg, i.v.; n = 7). Each point represents mean ± SEM percentage gating; auditory gating was calculated from 50 consecutive evoked potentials (^#p < 0.01 vs AMP; ^*p < 0.01 vs control). D, Dose-response effect of PNU-120596 administered subcutaneously on an amphetamine-induced gating deficit expressed as percentage of reversal (^#p < 0.05 vs AMP; ^*p < 0.05 vs vehicle; total of 46 animals included in dose-response). Error bars represent SEM.